CD4O and its ligand, gp39, were first thought only to be important in the regulation of thymus-dependent (TD) immunity, however, evidence from our lab and others have shown that the interactions of CD40 and gp39 can have a profound impact on cell mediated immunity. We have shown that a brief treatment with anti-gp39 can completely block the development of acute graft vs host disease (aGVHD). Therefore, we propose to use GVHD both as a model to understand the basic mechanisms of how cD40 and its ligand regulate the development of T cell reactivities to transplantation antigens and also as a clinically relevant model of graft rejection. We present a hypothesis that anti-gp39 blocks multiple immune mechanisms that mediate graft rejection. First. we propose that gp39 is critical in the inductive phase of T cell immunity to transplantation antigens. In a model of aGVHD, the preliminary data shows that anti-gp39 can diminish the magnitude of the CD4 and CD8 responses to alloantigen. We propose that this is due to the fact that host dendritic cells (DC) cannot adequately mature in the absence of a CD4O signal, We propose that gp39 is critical for the in vivo upregulation of co-stimulatory molecules, the migration of DC into appropriate anatomical sites to optimize T cell priming, as well as the production of cytokines (IL12) that control the course of T cell differentiation. Second, we propose that gp39 function impacts on the clonal expansion of alloreactive T cells and also, through its actions on antigen presenting cells (APC), plays a critical role in polarizing (Th1 vs. Th2) the T cell compartment. These studies will provide a fundamental appreciation for the role of CD4O in its regulation of APC maturation, a common theme of Projects #1 and #2. It is likely that a variety of CD4O-bearing cells impact on the development and pathogenesis of aGVHD. Therefore, our third aim is to gain an appreciation for the relative contribution of different CD40 bearing cells in the development of aGVHD. To achieve this goal, a human (h)CD4O transgene will be selectively reconstituted in specific cellular compartments in the CD4O-1-mouse. If it is true that gp39 contributes to the immune response to transplantation antigens at multiple levels, then the selective expression of CD4O in different compartments (B cell, DC, Macrophage, endothelial cell) should produce a distinctive phenotype. Finally, our fourth goal is to determine which biochemical pathways invoked by CD4O are critical to the development of GVHD. Over the past two years, a number of the signalling elements in the CD4O signalling cascade have been elucidated. Using strategies to interfere with CD4O signalling chains, we will address which of the CD4O elements are crucial for disease development. This approach will integrate the basic studies on the biochemistry of CD4O signalling (Project #4) and the in vivo function of CD4O in the regulating the immune response to transplantation antigens (Projects #1,2 &3).